Newcastle disease virus (NDV) causes a highly contagious and fatal disease affecting all species of birds. Newcastle disease can vary from mild to highly virulent depending upon the virus strain and the host species (1). The virus is a member of the family Paramyxoviridae (2) and contains a single-stranded negative-sense RNA genome.
The genome of NDV is a single strand negative-sense RNA, which has been founded to consist of 15,186 nucleotides (3). The genomic RNA contains six structural genes, which encode at least seven proteins (4,5). Three proteins constitute the nucleocapsid; specifically the nucleoprotein (NP), the phosphoprotein (P), and the large polymerase protein (L). Two proteins form the external envelope spikes, namely the F and HN proteins. The matrix protein (M) forms the inner layer of the virion. The genomic RNA is tightly bound by the NP protein and with the P and L proteins form the functional nucleocapsid within which resides the viral transcriptive and replicative activities. The HN glycoprotein is responsible for attachment of virus to host cell receptors and the F glycoprotein mediates fusion of the viral envelope with the host cell plasma membrane thereby enabling penetration of viral genome into cytoplasm (6). The HN and F proteins are the main targets for the immune response (7, 8). In common with several other Paramyxoviruses, NDV produces a seventh protein (V) of unknown function by editing of the P gene (5, 9).
NDV follows the general scheme of transcription and replication of other nonsegmented negative-strand RNA viruses. The polymerase enters the genome at a promoter in the 3′ extragenic leader region and proceeds along the entire length by a sequential stop-start mechanism during which the polymerase remains template bound and is guided by short consensus gene-start (GS) and gene-end (GE) signals. This generates a free leader RNA and six nonoverlapping subgenomic mRNAs. The abundance of the various mRNAs decreases with increasing gene distance from the promoter. The genes are separated by short intergenic regions (1-47 nucleotides) which are not copied into the individual mRNAs. The 3′ terminus (leader) and the 5′ terminus (trailer) of the genomic RNA contain the cis-acting sequences important for replication, transcription, and packaging of viral RNA (10). RNA replication occurs when the polymerase somehow switches to a readthrough mode in which the transcription signals are ignored. This produces a complete encapsulated positive-sense replicative intermediate which serves as the template for progeny genomes. A schematic of the genetic map of NDV genomic RNA is shown in FIG. 1.
Vaccination has been widely used to control Newcastle disease. The most commonly used method of vaccination has been the exposure of chickens to low virulence strains of NDV. Advantages of live Newcastle disease vaccines are that they can be mass-applied by natural routes of infection and that protection occurs very soon after application resulting in local as well as systemic immunity.
The main disadvantage of live Newcastle disease vaccines is that they can cause disease and can lead to mortality. Thus, development of a completely apathogenic NDV vaccine would be beneficial to the poultry industry. Before the present invention was made, there was no method available to directly manipulate the genome of NDV to achieve a desired level of attenuation.
A few years ago, two alternative approaches were developed for nonsegmented negative-stranded RNA viruses. In one approach, synthetic “minigenomes” consisting of genomic terminal sequences surrounding a reporter gene were transcribed from cDNA in vitro and transfected into cells infected with wild type helper virus. The second approach involved co-expression of minigenomes and necessary nucleocapsid proteins from transfected plasmids using the transient vaccinia virus/T7 RNA polymerase expression system. These approaches have made it possible to begin the characterization of cis- and trans-acting factors required for transcription and replication of several nonsegmented negative-stranded RNA viruses. Recently, the second approach was used to recover complete infectious recombinant virus from full-length cDNA for several nonsegmented negative-strand RNA viruses, namely, rabies virus, vesicular stomatitis virus, measles virus, Sendai virus, human respiratory syncytial virus, rinderpest virus and parainfluenza 3 virus.
Another major disadvantage of currently available NDV vaccines is that the avirulent vaccine viruses can change to virulent viruses by reversion of up to a few, e.g. one or two, nucleotides at the cleavage site. For instance, currently available NDV vaccines that are of low virulence strains differ from virulent strains by only one or two nucleotides in the F0 protein cleavage site, which is fifteen nucleotides long. Therefore, reversion of these few nucleotides can change the phenotype of the NDV from low virulence to highly virulence. Recently, it was shown that an outbreak of Newcastle disease in Australia was caused by a mutation in the cleavage site on F0 protein of pre-existing avirulent field strains circulatory in eastern Australia (XI International Congress of Virology Abstracts, VET.06, pp. 102).